1. Field of the Invention
The present invention is a method and apparatus that provides an inexpensive, quick, and portable means to separate motile sperm from a sperm sample. The invention passively filters the motile sperm using a nucleopore membrane.
2. Background Art
Artificial insemination has become a much more frequent procedure due to a variety of sociological, economic, and perhaps environmental reasons. The number of inseminations in the United States and worldwide has been increasing, and will likely continue to increase in the next millennium. A number of causes have been cited for this increase. The age of childbearing has increased, as people are delaying children and marriage. The increased age lowers the probability of fertilization for both men and women. More and more women are also raising children by themselves without a partner, and electing artificial insemination as a means of fertilization. In addition, those that would otherwise not be able to have children now have a medical option that is relatively affordable. An additional factor may be that the sperm count among males has been declining, making fertilization more difficult. And finally, environmental factors have been blamed for decreased fertility of both men and woman.
There are currently a wide array of artificial insemination methods such as, intracervical, intrauterine (IUI), intratubular and direct intraperitoneal (DIPI) insemination, gamete intrafallopian transfer (GIFT), in vitro fertilization and embryo transfer (IVFET), zygote intrafallopian transfer (such as ZIFT, PROST and TET), peritoneal oocyte and sperm transfer (POST), and sex selection, among others. As technology advances, other methods are certain to follow, however, regardless of the process, high motile sperm are preferred.
As an example, the Intrauterine Insemination (IUI) and In Vitro fertilization (IVF) methods attempt to recreate the reproductive process by placing sperm and eggs together in an environment conducive to fertilization either in the womb or outside the womb. The fertilization process requires the sperm to actively invade the egg and commence fertilization. Motile sperm are much more likely to penetrate the egg.
A typical semen sample contains materials such as paternal plasma, protein, leukocytes, spermdecapitation factors and other extraneous materials, and dead, agglutinated or nonviable spermatozoa. These materials are known to interfere with successful fertilization, and with the successful maintenance of a fertilized ovum in the female patient. As an example, seminal plasma can cause severe uterine cramping, and in a worst case result in spontaneous abortion of the fertilized ovum. Thus, it is desirable to remove those spermatozoa having relatively low motility or possessing unhealthy, damaged or abnormal membranes.
The total number of sperm in an ejaculate is a measure of fertility, however, the percentage of motile sperm is much more important, especially when considering alternate reproductive means.
Sperm are categorized according to the their exhibited motility, as exhibited by Table 1, below.
TABLE 1 ______________________________________ Motility Index Degree of Motility Type of Motility ______________________________________ 0 No motility, or movement of tail with no forward progression 1 20% or less showing forward progression (sluggish movement) 2 20%-50% showing forward progression 3 50%-80% showing forward progression 4 80%-100% showing forward progression (very rapid movement) ______________________________________
The percentage or motile sperm showing progressive swimming movements is a measure of the fertility of the sperm sample. The higher the percentage, the higher quality of the sperm sample, and the greater the likelihood that the sample will achieve fertilization.
A high quality sperm sample is important for many reasons. The process of artificial insemination is not only costly economically, but is psychologically expensive. Unsuccessful attempts have devastating effects on the patients.
Higher quality sperm samples are also important considerations when the sample is subject to freezing or aqueous dilution, because these processes tend to kill or weaken the sample. Thus, only the highest quality sperm may survive the processing procedures to which the sperm are subjected.
In summary, higher viability spermatozoa are more likely to lead to successful fertilization and impregnation. The higher quality sperm are also more likely to survive freezing (cryopreservation) and other processing procedures.
In order to reduce the aforementioned problems, attempts have been made to provide a simple, portable, and inexpensive method and device for the filtration of sperm. However, the prior art devices and methods had significant disadvantages and shortcomings.
Various methods of selecting the more active sperm have been utilized in the past, such as the swim up. Swim down and Percoil density gradient centrifugation techniques. Swim-up methods are commonly used to process fresh or frozen specimens for the IUI and IVF procedures. The sperm is placed in a medium and subjected to a centrifuge process. The more motile sperm swim to a level where they can be extracted. Such methods employ multiple tube and centrifugation steps that are time consuming and can lead to a low recovery of motile sperm.
Table 2 below, provides a comparison of some features of these methods.
TABLE 2 ______________________________________ Characteristics of Prior Art Methods Feature Swim-Up Strain Ex Percoll Enhance-S Plus ______________________________________ Sperm None None 1 1 wash/ Preparation wash/centri- centrifugation after fugation liquefaction Preparation Prepare None Build a 2 Build a 2 step of device buffer solu- step gradient gradient tion, pour into plate Sperm Quality Motility Higher % Higher % Same Same motility motile motile motility Viability unknown unknown unknown unknown Leukocyte unknown unknown unknown unknown contaminants Washes re- None None 1-2 1-2 quired after separation Total # 1 1 2-3 2-3 Centrifu- gation Endotoxin No No Yes Yes Contamina- tion ______________________________________
One prior art device used for separating the motile sperm from a sperm sample is the migration-sedimentation chamber. This system uses a culture medium with the sperm sample suspended in the medium, and requires a centrifuge to rapidly spin the chamber to separate the motile sperm. And, because it is an active process, it increases the likelihood that motile sperm will be excluded from the sample set, or damaged during the process, thus producing a lower recovery sample.
In operation, the sperm sample is placed in a migration-sedimentation chamber, preferably in the lower well portion. The chamber is subjected to centrifugation, and the re-suspended sperm pellet is transferred to the gallery segment of the chamber. The portion of sperm in the well is the motile portion, and is used for insemination.
U.S. Pat. No. 5,185,246 ('246) is a method for semen analysis employing a membrane separation. This patent discloses a method to separate the particulate (cells, spermatozoa, and other particulate) in the whole semen from the seminal plasma which contains many soluble compounds, proteins, hormones, small molecules and electrolytes. The pore size is specifically designed for such a separation, and all sperm cells are blocked from passing through the membrane. The driving force of the '246 patent is a vacuum, and the sperm cells are stuck to the filter, dead and immobile, amenable only for in-situ testing or further disruption. A specific volume, preferably 100 microliters with various aqueous dilutions is employed. The separation apparatus of the '246 patent is a well filter plate that is used for multiple testing in the same round of processing.
The '246 patent is related to tests of andrological significance that need be performed on either the seminal plasma (in the absence of sperm cells) or on the surface of the sperm cells (in the absence of the seminal plasma). But, the cells in these cases need not be viable, need not be motile, and need not be freely dispersed in an aqueous buffered medium for retrieval. The '246 patent discloses a device for providing easier diagnostic access to specific components of the semen sample.
Another such invention is described in U.S. Pat. No. 5,575,914, which discloses a conduit that has glass wool as a strainer mechanism. The glass wool is compressed to a density sufficient to permit passage of the more motile sperm while blocking the lesser or non-motile sperm. In the preferred embodiment, the conduit is a nested pair of tubes that try to keep the glass wool density consistent.
The invention of U.S. Pat. No. 4,009,260 describes the fractionation of sperm through layered migration of different layers having differing densities. The more motile sperm penetrate deeper into the layers. Similarly, U.S. Pat. No. 4,007,087 also discloses the fractionation via layered solutions contacting the sperm sample.
U.S. Pat. No. 5,427,946 discloses a channeling apparatus, where there are inlet ports, flow channels, and nesting chambers. The sperm sample is applied at the inlet port, and only the motile sperm are capable of reaching the chambers.
In summary, problems heretofore exhibited in the art include having to purchase and maintain additional equipment such as a centrifuge. Prior methods required taking trips to other sites to perform the separation or centrifuge process. Other methods required extensive time to separate the motile sperm. In addition, prior art methods that used active separation schemes removed motile sperm and reduced the recovery lot.
What is needed is a method and apparatus that can passively isolate higher viability spermatozoa from the spermatozoa having relatively lower motility. The device should be inexpensive, portable, and easy-to-use. The method should be sufficient to screen out the lesser sperm and other compounds, leaving only high quality sperm for insemination. What is needed is an efficient device and method of removing the less motile sperm without costly or time-consuming procedures, and this new system should provide a quick and inexpensive way to increase the likelihood of fertilization.